Method for silk fibroin gelation using sonication

ABSTRACT

This invention provides for a process of rapidly forming silk fibroin gelation through ultrasonication. Under the appropriate conditions, gelation can be controlled to occur within two hours after the ultrasonication treatment. Biological materials, including viable cells, or therapeutic agents can be encapsulated in the hydrogels formed from the process and be used as delivery vehicles.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. §371 National Stage of InternationalApplication No. PCT/US2008/065076 filed on May 29, 2008, whichdesignates the United States, and which claims the benefit of priorityunder 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/940,554,filed May 29, 2007, the contents of which are herein incorporated byreference in its entirety.

This invention was made with U.S. government support under TissueEngineering Research Center Grant No. P41 EB002520 awarded by theNational Institutes of Health. The government has certain rights in theinvention.

FIELD OF THE INVENTION

This invention provides for methods of rapidly forming silk fibroingelation through ultrasonication. The hydrogels formed from the methodare useful, for example, as biodelivery vehicles.

BACKGROUND

Biocompatible and biodegradable polymer hydrogels are useful carriers todeliver bioactive molecules and cells for biomedical applications, suchas in tissue engineering and controlled drug release. Purified nativesilk fibroin forms β-sheet-rich crosslinked hydrogel structures fromaqueous solution, with the details of the process and gel propertiesinfluenced by environmental parameters. Previous gelation times oftentook days to weeks for aqueous native silk protein solutions, with hightemperature and low pH responsible for increasing gelation kinetics.Those conditions, although suitable for incorporation of some bioactivemolecules, may be too slow for incorporation of active cell and labilebioactive molecules.

Thus, there is a need in the art for a process of rapidly forming silkfibroin gelation at mild physiological conditions.

SUMMARY OF THE INVENTION

This invention relates to a process of rapidly forming silk fibroingelation. The process exposes silk fibroin to a treatment comprisingultrasonication for a sufficient period of time to initiate gelation.For example, under particular conditions the gelation occurs within 24hours of the ultrasonication treatment.

An embodiment of the invention also relates to a method of controllinggelation time of silk fibroin by contacting a silk fibroin solution withan ultrasonication treatment for a sufficient period of time to initiategelation. In one example the gelation time is under two hours.

Another embodiment relates to a method of encapsulating an agent in silkfibroin. The method comprises exposing a silk fibroin solution to anultrasonication treatment for a period of time to initiate gelation, andintroducing the agent to the silk fibroin solution before substantialgelation occurs in the silk fibroin solution, thereby forming asilk-fibroin-encapsulated agent. Alternatively, the agent may be addedto the silk fibroin before sonication. The agent can be a therapeuticagent, such as a drug, or a biological material, such as a cell. Forexample, human bone marrow derived mesenchymal stem cells (hMSCs) weresuccessfully incorporated into silk fibroin hydrogels after sonication,followed by rapid gelation and sustained cell function.

The hydrogels resulting from the methods of the invention exhibit bothgood mechanical properties and proteolytic degradation profiles. Forexample, sonicated silk fibroin solutions at 4%, 8%, and 12% (w/v),followed by adding hMSCs, gelled within 0.5 hours to 2 hours. The cellsgrew and proliferated in the 4% gels over twenty-one days. Additionally,low concentrations of K⁺ and low pH may be used to promote gelation.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts silk fibroin (SF) gelation under various sonicationconditions. 0.5 ml of aqueous solution was used, sonication wasperformed at 20% amplitude and time varied from 5 sec to 30 sec. Valuesare average±standard deviation of a minimum of N=3 samples for eachgroup. *Significant differences between the groups (Student's t-test,p<0.01).

FIGS. 2A-2C depict the dynamic silk β-sheet structure formation duringthe gelation process. FIG. 2A shows Circular Dichroism (CD) measurementson sonicated 2% (w/v) silk fibroin aqueous solutions with wavelengthscans taken every 8 min after sonication for 120 min. FIG. 2B shows achart of ellipticity increase at 217 nm (β-sheet structure peak)recorded against time. FIG. 2C is a schematic illustration of mechanismof silk gelation. The gelation process contains two kinetic steps (a)structural change from random coil to β-sheet with some inter-chainphysical cross-links occurring in a short time frame; (b)β-sheetstructure extended, large quantity of inter-chain β-sheet cross-linksformed, and molecules organized to gel network over a relatively longtime frame.

FIGS. 3A-3C show salt and pH effects on silk fibroin gelation. Prior tosonication, solutions at various concentrations were supplemented withK⁺ (FIG. 3A) and Ca²⁺ (FIG. 3B) to final concentrations of 20 mM-200 mM.FIG. 3C shows the effects of adjusting the pH of the silk fibroinaqueous solution prior to sonication. Sonication was performed at 20%amplitude for 15 sec for all samples. Values are average±standarddeviation of a minimum of N=3 samples for each group. *, ⋄ Significantdifferences between the groups (Student's t-test, p<0.05).

FIG. 4A-4C present charts analyzing the mechanical properties of silkfibroin hydrogels. The top two charts show published results fromnon-sonicated hydrogels, and the bottom two charts showsonication-processed hydrogels run in accordance with the invention. Thetwo charts on the left show the effects of compressive strength, and thetwo charts on the right show the effects of compressive modulus. Thehydrogels prepared from silk fibroin aqueous solutions (the two topcharts) were run at various temperatures, the hydrogels that weresonicated (two bottom charts) were run at various sonication treatments.Values are average±standard derivation of a minimum of N=3 samples.

FIG. 5 depicts the enzymatic degradation of silk fibroin hydrogels.Hydrogels at 4%, 8%, and 12% (w/v) were prepared by sonication andimmersed in either PBS, pH 7.4 (control) or protease XIV in PBS (5 U/ml)for seven days. The mass remaining was determined by comparing the wetweight of gel plugs at each time point with original wet weight. Valuesare average±standard derivation of a minimum of N=4 samples.

FIG. 6 depicts graphically DNA quantification of hMSCs encapsulated insilk fibroin hydrogels. DNA content in each gel group was analyzed withPicoGreen assay, and the results were normalized by the wet weight ofeach gel plug. Values are average±standard derivation of a minimum ofN=4 samples. *Significant differences between the groups (Student'st-test, p<0.05).

DETAILED DESCRIPTION

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

As used herein and in the claims, the singular forms include the pluralreference and vice versa unless the context clearly indicates otherwise.Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.”

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood to one of ordinaryskill in the art to which this invention pertains. Although any knownmethods, devices, and materials may be used in the practice or testingof the invention, the methods, devices, and materials in this regard aredescribed herein.

This invention relates to a process of rapidly forming silk fibroingelation. The process exposes silk fibroin to a treatment comprisingultrasonication for a sufficient period of time to initiate gelation.This approach provides for ultrasonication-based methods used toaccelerate the sol-gel transition in a temporally controllable manner.Gelation time can be controlled from minutes to hours based on thesonication parameters used (energy output, duration time, and others)and silk fibroin concentration within physiologically relevantconditions. After sonication, the silk fibroin undergoes a rapidstructural change from random coil to β-sheet, corresponding togelation. An agent can be added, for example a therapeutic agent or abiological agent, either before, during or after the sonicationtreatment, and encapsulated upon gelation. The present invention thusprovides for methods useful for various biomedical applications, such asthose in which the encapsulation of cells is time sensitive.

Hydrogels are considered useful scaffolds for encapsulation and deliveryof cells and bioactive molecules, such as for tissue engineering andcell therapeutic applications, due to their high water content;usually >30% (Park & Lakes, BIOMATS: INTRO. (2nd ed., Plenum Press, NY,1992). Hydrogels used in these types of applications have mechanical andstructural properties similar to some tissues and extracellular matrices(ECM), therefore, they can be implanted for tissue restoration or localrelease of therapeutic factors. To encapsulate and deliver cells,hydrogels should, preferably, be formed without damaging cells, benontoxic to the cells and the surrounding tissue, be biocompatible, havesuitable mass transport capability to allow diffusion of nutrients andmetabolites, have sufficient mechanical integrity and strength towithstand manipulations associated with implantation, have controllablelifetimes, and should maintain gel volume after implantation for areasonable period of time depending on the application (Drury & Mooney,24 Biomats. 4337-51 (2003).

A variety of synthetic materials, such as poly(ethylene oxide) (PEO),poly(vinyl alcohol) (PVA), poly(acrylic acid) (PAA), polypropylenefurmarate-co-ethylene glycol) (P(PF-co-EG)), and naturally derivedmaterials, such as agarose, alginate, chitosan, collagen, fibrin,gelatin, and hyaluronic acid (HA) have been used to form hydrogels.Gelation occurs when the polymer chains crosslink either chemically orphysically into networks, triggered by chemical reagents (e.g.,cross-linkers) or physical stimulants (e.g., pH and/or temperature).Hydrogels formed from synthetic polymers offer the benefit of gelationand gel properties that are controllable and reproducible, through theuse of specific molecular weights, block structures, and crosslinkingmodes. Generally, gelation of naturally derived polymers is lesscontrollable, although they tend to be useful as carriers of cell andbioactive molecules for tissue engineering and implantable medicaldevices because their macromolecular properties are more closely alignedto the extracellular matrix and the degradation products are nontoxic(Lee et al., 221 Int'l J. Pharma. 1-22 (2001); Smidsrød et al., 8 TrendsBiotech. 71-78 (1990).

Among naturally derived biomaterials, silk fibroin protein, theself-assembling structural protein in natural silkworm fibers, has beenstudied because of its excellent mechanical properties,biocompatibility, controllable degradation rates, and inducibleformation of crystalline β-sheet structure networks (Altman et al., 24Biomats. 401-16 (2003); Jin & Kaplan, 424 Nature 1057-61 (2003); Horanet al., 26 Biomats. 3385-93 (2005); Kim et al., 26 Biomats. 2775-85(2005); Ishida et al., 23 Macromolecules 88-94 (1990); Nazarov et al., 5Biomacromolecules 718-26 (2004)). Silk fibroin has been fabricated intovarious material formats including films, three dimensional porousscaffolds, electrospun fibers and microspheres for both tissueengineering and controlled drug release applications (Jin et al., 5Biomacromolecules 711-7 (2004); Jin et al., 3 Biomacro-molecules,1233-39 (2002); Hino et al., 266 J. Colloid Interface Sci. 68-73 (2003);Wang et al., 117 J. Control Release, 360-70 (2007)). See also U.S.patent application Ser. No. 11/020,650; No. 10/541,182; No. 11/407,373;and No. 11/664,234; PCT/US07/020,789; PCT/US08/55072.

In nature, silk fibroin aqueous solution is produced in the posteriorsection of silkworm gland and then stored in the middle section at aconcentration up to 30% (w/v) and contains a high content of random coilor alpha helical structure. During fiber spinning into air, high shearforce and elongational flow induces self-assembly and a structuraltransition to the β-sheet structure, leading to the formation of solidfibers (Vollrath & Knight, 410 Nature, 541-48 (2001)). The presence ofmetallic ions and pH changes in different sections of the glandinfluence this transition (Chen et al., 3 Biomacromolecules 644-8(2002); Zhou et al., 109 J. Phys. Chem. B 16937-45 (2005); Dicko et al.,5 Biomacromolecules 704-10 (2004); Terry et al., 5 Biomacromolecules768-72 (2004)). In vitro, purified silk fibroin aqueous solutionsundergo self-assembly into β-sheet structures and form hydrogels. Thissol-gel transition is influenced by temperature, pH, and ionic strength(Wang et al., 36 Int'l J. Biol. Macromol. 66-70 (2005); Kim et al., 5Biomacromolecules 786-92 (2004); Matsumoto et al., 110 J. Phys. Chem. B21630-38 (2006)). The compressive strength and modulus of silk hydrogelsincreases with an increase in silk fibroin concentration and temperature(Kim et al., 2004).

Silk fibroin hydrogels are of interest for many biomedical applications.For example, fibroin hydrogels were used as a bone-filling biomaterialto heal critical-size cancellous defects of rabbit distal femurs, wherethe silk gels showed better bone healing than the poly(D,Llactide-glycolide) control material (Fini et al., 26 Biomats. 3527-36(2005)).

For many cell-based applications, gelation must be induced under mildconditions in a relatively short period of time (within hours). Silkgelation time may be prohibitively long, however, unlessnonphysiological treatments are considered (such as low pH, hightemperature, additives) in the absence of chemical modifications to thenative silk fibroin protein. For silk fibroin concentrations from 0.6%to 15% (w/v), days to weeks were required for the sol-gel transition atroom temperature or 37° C. (Kim et al., 2004; Matsumoto et al., 2006;Fini et al., 2005)). Adding salts at concentrations above physiologicallevels does not significantly alter the gelation kinetics (Kim et al.,2004). Lowering pH (pH<5) or increasing temperature (>60° C.) couldreduce the gelation time to a few hours (Kim et al., 2004; Fini et al.,2005; Motta et al., 15 J. Biomater. Sci. Polymer. Edu. 851-64 (2004)),but these conditions could potentially alter cell function and affectcell viability.

In the present invention, novel methods to accelerate the process andcontrol silk fibroin gelation are accomplished through ultrasonication.More specifically, a new ultrasonication-based method is presented thataccelerates the sol-gel transition in a temporally controllable manner.Mechanistically, the process induces physical β-sheet crosslinking viaalteration in hydrophobic hydration of the fibroin protein chains. Thispermits cell additions post-sonication, followed by rapid gelation.Gelation time may be controlled from minutes to hours based on thesonication parameters used (energy output and duration time) and silkfibroin concentrations. The method further provides for manipulation ofthe pH and salt concentration effects on gelation; the dynamic silkstructural changes after gelation; and the behavior of encapsulatedcells, such as human bone marrow derived mesenchymal stem cells (hMSCs)in silk gels.

Any type of silk fibroin may be used according to the present invention.Silk fibroin produced by silkworms, such as Bombyx mori, is the mostcommon and represents an earth-friendly, renewable resource. Organicsilkworm cocoons are commercially available. There are many differentsilks, however, including spider silk, transgenic silks, geneticallyengineered silks, and variants thereof, that may be used alternatively.An aqueous silk fibroin solution may be prepared from the silkwormcocoons using techniques known in the art. Suitable processes forpreparing silk fibroin solution are disclosed in, for example, U.S.patent application Ser. No. 11/247,358; WO/2005/012606; andPCT/US07/83605. For instance, silk used in a silk biopolymer may beattained by extracting sericin from the cocoons of B. mori.

Substantial gelation usually occurs within twenty-four hours after theultrasonication treatment. For example, the silk fibroin gel forms lessthan four hours after ultrasonication treatment, such as within twohours after the ultrasonication treatment. In a particular embodiment,the silk fibroin undergoes gelation at a time period ranging from aboutfive minutes to about two hours after the ultrasonication treatment.Thus, depending on requirements, gelation time can occur from minutes tohours, based on the ultrasonication treatment used in the preparation ofthe solution.

Ultrasonication treatments are known in the art. For the purposes ofthis application, the terms “ultrasonication” and “sonication” are beingused interchangeably and carry the same meaning. Ultrasonicationtreatments may be performed in any manner known in the art that appliesultrasonication to the silk fibroin. The ultrasonication treatment mayinvolve exposing the silk fibroin to sonication one time, or may involvemultiple separate exposures. Sonication has been studied in the contextof protein structural changes (Meinel et al., 71 J. Biomed. Mater. Res.A 25-34 (2004); Meinel et al., 88 Biotechnol. Bioeng. 379-91 (2004)) andhas been used to generate large liquid-gas interfaces, local heatingeffects, mechanical/shear stresses, and free radical reactions. Incontrast, in other studies relating to peptide gelation, the assembledpeptide nanofibers in the gel were disrupted into smaller fragments bysonication (Hung et al., 32 Ann. Biomed. Eng. 35-49 (2004)). In thecontext of polymer sol-gel transitions, sonication has typically beenused to break down gel networks and reliquify hydrogels. The presentinvention provides for the novel use of sonication to induce silksol-gel transition.

The ultrasonication treatment should last for a period of timesufficient to initiate the gelation process, but not so long as tocompromise the mechanical properties of the hydrogel. Typically,ultrasonication treatments may last from about 5 seconds to about 60seconds, depending on the amount of silk fibroin used, the concentrationof the solution, and other factors appreciated by those of ordinaryskill in the art. For example, the ultrasonication treatments last fromabout 15 seconds to about 45 seconds. Gelation typically begins at theonset of the ultrasonication treatment and continues after the treatmentends.

The ultrasonication treatment may include other treatments to assist inthe gelation process. For example, the treatment may include a saltsolution. Salt solutions are known in the art to assist in inducinggelation. Typical salt solutions containing ions of potassium, calcium,sodium, magnesium, copper, and/or zinc may be used. Potassium may beadvantageous in a salt solution in this context.

The treatment can also include adjusting the pH of the aqueous fibroinsolution. As known in the art, adjusting the pH of the aqueous solutioncan assist in inducing gelation. In particular, adjusting the pH eitherhigher or lower can be effective. Thus, for example, an aqueous solutionhaving a pH of about pH 4 or lower, or about pH 7.5 or higher, may beused.

In particular, using a potassium salt solution at low concentrations andat a low pH is often effective. A particular embodiment is directedtowards the use of a potassium salt where the salt concentration is lessthan 100 mM and the pH of the solution is about pH 4 or lower.

The invention also provides for a method of controlling gelation time ofsilk fibroin by contacting a silk fibroin solution with anultrasonication treatment for a sufficient period of time to initiategelation under conditions that gelation occurs within about two hours.The sonication process results in interactions among the silk fibroinchains. A particular embodiment provides for a method of controllinggelation time so that the silk fibroin undergoes gelation at a timeperiod ranging from about five minutes to about two hours after theultrasonication treatment.

Additionally, various other factors can be used to control the gelationtime. For example, the gelation time can be controlled through theamplitude of the ultrasonication and the concentration of the silkfibroin solution. For example, the amplitude ranges from about 25% toabout 35% power output (typically, 7 watts to 10 watts) and theconcentration of the silk fibroin ranges from about 10% to about 15%(w/v). In another embodiment, the amplitude ranges from about 25% toabout 55% power output (typically, 7 watts to 21 watts) and theconcentration of the silk fibroin ranges from about 5% to about 10%(w/v). Those of ordinary skill in the art, in light of the presentapplication, are able to alter the amplitude of the ultrasonication andthe concentration of the silk fibroin solution to produce the desiredlevel of gelation and the desired time frame in which gelation occurs.

The gelation time may also be controlled by adding a salt solution andadjusting the concentration of the silk fibroin solution and theconcentration of the salt solution. The salt solution may includepotassium ions, but other salt solutions may be used. In a specificembodiment, the concentration of the silk fibroin is 4% (w/v) or lower,and the concentration of the potassium salt solution ranges from 20 mMto 100 mM.

Additionally, gelation time may be controlled by adjusting theconcentration and pH of the salt solution, especially when the saltsolution contains potassium ions. In a particular embodiment, the saltsolution is a potassium salt solution at a pH of about pH 4 or lower.For example, the potassium salt solution has a concentration of 20 mM to100 mM.

The invention also relates to a method of encapsulating at least oneagent in silk fibroin. The method comprises (a) exposing a silk fibroinsolution with an ultrasonication treatment for a period of time toinitiate gelation; and (b) introducing the agent into the silk fibroinbefore substantial gelation occurs in the silk fibroin, thus forming asilk-fibroin encapsulated agent. The agent may be introduced into thesilk fibroin solution before, during, or after the ultrasonicationtreatment.

The agent can represent any material capable of being encapsulated inthe silk fibroin gel. For example, the agent may be a therapeutic agent,such as small molecules and drugs, or a biological material, such ascells (including stem cells), proteins, peptides, nucleic acids (DNA,RNA, siRNA), PNA, aptamers, antibodies, hormones, growth factors,cytokines, or enzymes. Encapsulating either a therapeutic agent orbiological material is desirous because the encapsulated product can beused for biomedical purposes.

If a therapeutic agent is being encapsulated, the therapeutic agent canbe introduced into the silk fibroin solution before, during, or afterthe ultrasonication treatment, as most therapeutic agents are notaffected adversely by sonication. On the other hand, if a biologicalmaterial is being encapsulated, the biological material may be affectedadversely by the sonication and should typically not be introduced intothe silk fibroin solution until after the ultrasonication treatment.This may not be necessary for all biological material, but sonicationhas been known to damage or destroy living cells, so caution may beapplied.

When an agent is introduced after the ultrasonication treatment, theconditions of the ultrasonication treatment may be adjusted so thatgelation occurs some period of time after the ultrasonication treatment.If gelation occurs during the ultrasonication treatment or immediatelythereafter, an insufficient amount of time may exist to introduce theagent into the silk fibroin solution. For example, when the agent isintroduced after the ultrasonication treatment, the silk fibroinundergoes gelation at a time period ranging from about five minutes toabout two hours after the ultrasonication treatment.

If an agent is introduced before or during the ultrasonicationtreatment, gelation can occur during the ultrasonication treatment,immediately thereafter, or a period of time after the ultrasonicationtreatment. Therefore when the agent is introduced before or during theultrasonication treatment, the silk fibroin may undergo gelation withinabout two hours after the ultrasonication treatment.

When introducing therapeutic agents or biological material into the silkfibroin, other materials known in the art may also be added with theagent. For instance, it may be desirable to add materials to promote thegrowth of the agent (for biological materials), promote thefunctionality of the agent after it is released from the encapsulation,or increase the agent's ability to survive or retain its efficacy duringthe encapsulation period. Materials known to promote cell growth includecell growth media, such as Dulbecco's Modified Eagle Medium (DMEM),fetal bovine serum (FBS), non-essential amino acids and antibiotics, andgrowth and morphogenic factors such as fibroblast growth factor (FGF),transforming growth factors (TGFs), vascular endothelial growth factor(VEGF), epidermal growth factor (EGF), insulin-like growth factor(IGF-I), bone morphogenetic growth factors (BMPs), nerve growth factors,and related proteins may be used. Additional options for delivery viathe gels include DNA, siRNA, antisense, plasmids, liposomes and relatedsystems for delivery of genetic materials; peptides and proteins toactive cellular signaling cascades; peptides and proteins to promotemineralization or related events from cells; adhesion peptides andproteins to improve gel-tissue interfaces; antimicrobial peptides; andproteins and related compounds.

The silk-fibroin encapsulated therapeutic agents or biological materialare suitable for a biodelivery device. Techniques for using silk fibroinas a biodelivery device may be found, for example, in U.S. patentapplication Ser. No. 10/541,182; No. 11/628,930; No. 11/664,234; No.11/407,373; PCT/US207/020789; PCT/US08/55072.

The silk fibroin hydrogel structure enables the biodelivery vehicle tohave a controlled release. Controlled release permits dosages to beadministered over time, with controlled release kinetics. In someinstances, delivery of the therapeutic agent or biological material iscontinuous to the site where treatment is needed, for example, overseveral weeks. Controlled release over time, for example, over severaldays or weeks, or longer, permits continuous delivery of the therapeuticagent or biological material to obtain preferred treatments. Thecontrolled delivery vehicle is advantageous because it protects thetherapeutic agent or biological material from degradation in vivo inbody fluids and tissue, for example, by proteases.

Further regarding the approach to inducing silk gel formation usingsonication, samples of 0.5 mL silk fibroin aqueous solutions atconcentrations of 1%, 2%, 6%, and 20% (w/v) were sonicated as describedbelow. When power output was kept constant (20% amplitude), silk fibroingelation time decreased with increased sonication time (FIG. 1). Forevery increase in silk concentration from 1% to 6% (w/v), the gelationtime decreased significantly (p<0.01 between * samples in FIG. 1). The20% (w/v) sample had a similar or even longer gelation time than the 6%(w/v) sample (FIG. 1). This outcome for the 20% sample is likely due tothe high viscosity of the solution, thus sonication waves could noteffectively propagate in the solution. When the power output above 30%amplitude was used, sonication generated thick foams and the silkfibroin did not gel in a homogeneous manner.

This foaming was not observed when the volume for sonication wasincreased to 5 ml, even at power levels as high as 55% amplitude. Whenhigher concentrations were sonicated at volumes exceeding 5 ml, however,heterogeneous gelation occurred. Small volumes of silk solution (withoutautoclaving) were used for sonication optimization and gelcharacterizations (pH, salt effect, and CD measurement), and autoclavedsilk solutions were used for mechanical, degradation, and cellencapsulation studies. Interestingly, when compared with the originalsolutions, autoclaving did not significantly change the sonicationparameters used and the related gelation times, suggesting that silkfibroin protein retained important features of its originalsolution-state structure and capability of structural transition toβ-sheet state in forming a gel after autoclave. Structural alterationsdue to autoclave treatments may be investigated further, but this aspectprovides for ease in commercial-scale preparation of pharmaceuticalproducts.

During gelation of silk fibroin, the sol-gel transition was linked to anincrease in β-sheet formation by observed changes in CD measurements(FIG. 2A). After sonication, rapid formation of β-sheet structure wasobserved, followed by a slower transition, based on the increase ofellipticity at 217 nm (FIG. 2B). Silk fibroin gelation occurred at thistransition point, where the initial rapid formation of β-sheet structureslowed. This transition is consistent with studies previously undertaken(Matsumoto et al., 2006), suggesting that similar mechanisms may beinvolved. The formation of β-sheet structure results from alteredhydrophobic interaction and the subsequent physical cross-links. Thisinitial step is followed by slower organization of the chains andformation of a gel network within a relatively long timeframe comparedwith the initial sonication-induced changes. This two-step silk gelationmechanism is schematically depicted in FIG. 2C.

The parameters studied to influence rates of gelation can be viewed as amethod to recapitulate the natural silkworm spinning process. The keyprocessing parameters include sonication effects, as a mimic forincreased shear forces experienced at the anterior division of thesilkworm gland, cation type and concentrations, and pH.

It is accepted that, in sonication, mechanical vibration causes theformation and collapse of bubbles. As a result of this cavitation, themedia may experience extreme local effects: heating (10,000 K), highpressure (200 bar) and high strain rates (10⁷ s⁻¹) (Paulusse & Sijbesma,44 J. Polym. Sci.-Polym. Chem. 5445-53 (2006); Kemmere et al., 290Macromol. Mater. Eng. 302-10 (2005). These physical phenomena have beenexploited in a variety of applications, including self-assembly andgelation of N-isopropylacrylamide/acrylic acid copolymer (Seida et al.,90 J. Appl. Polym. Sci. 2449-52 (2003)), organic fluids with metalatedpeptides (Isozaki 119 Angew Chem. 2913-15 (2007)), and syntheticself-assembling peptides (Yokoi et al., 102 Proc Nat Acad Sci USA8414-19 (2005)). Aside from peptides, proteins such as human serumalbumin and myoglobin have been studied with sonication as an approachto characterize aggregation and self-assembly related to disease states(Stathopulos et al., 13 Protein Sci. 3017-27 (2004); Mason & Peters,PRACTICAL SONOCHEM: USES & APPL. ULTRASOUND (2nd ed., Chichester, WestSussex, UK (2002)).

Given the breadth of behavior of polymer systems in response tosonication, it is likely that several physical factors related tosonication, including local temperature increases, mechanical/shearforces, and increased air-liquid interfaces affect the process of rapidgelation of silk fibroin. In particular, sonication-induced changes inhydrophobic hydration would result in the accelerated formation ofphysical cross-links, such as initial chain interactions related toβ-sheet formation. In the present study, during the sonication process,the solution temperature increased from room temperature to 40° C.-71°C. for the short period of time (5 min-6 min), which reflects atransient spike in local temperature. In a past study, gelation requireda few days when bulk samples were maintained at 60° C., withoutsonication (Kim et al., 2004). Therefore, local temperature effectslikely contribute toward the increased gelation kinetics, but are notsolely responsible for the short duration responses found. Localizedchain dynamics and changes in hydration states of the hydrophobicchains, influenced by the transient temperature increase, are likelyresponsible for the formation of the hydrophobic physical cross-links.

The unique hydrophobic block sequence features in silk fibroin chainsare particularly suitable for this type of technique due to the criticalrole of water in the control of intra- and inter-chain interactions (Jinet al., 2003). It might be useful to extend the technique to otherbiopolymer systems to determine the impact of chain chemistry onsonication controlled processes of chain assembly. Sonication relatedcollagen degradation, as a method to fragment chains to facilitatestudies of reassembly, have been reported (Giraud-Guille & Besseau, 113J. Struct. Biol. 99-106 (1994)). It should be noted that in the presentapproach did not result in significant chain degradation due to theshort duration sonication process used, based on SDS-PAGE analysis.

Silk fibroin aqueous solutions were supplemented with K⁺ and Ca²⁺ tovarious physiologically relevant concentrations prior to sonication. Asshown in FIG. 3A, at low K⁺ concentration (20 mM-50 mM), gelation timesignificantly decreased with increase in K⁺ concentration (p<0.05between * samples). At high K⁺ concentration (100 mM-200 mM), however,gelation was inhibited (FIG. 3A). These outcomes were observed for silkfibroin concentrations ranging from 0.5% to 8% (w/v). Above 8%, no salteffect was observed as gelation occurred fast in all the samples (<2min). Compared with K⁺, Ca²⁺ at the same concentrations induced slowersilk fibroin gelation (compare FIGS. 3A and 3B). When Ca²⁺ concentrationwas increased from 20 mM to 200 mM, silk fibroin gelation timesignificantly decreased (p<0.05 between * samples in FIG. 3B). Incontrast, in previous work, Ca²⁺ promoted silk fibroin gelation while K⁺had no effect (Kim et al., 2004), a different outcome than theobservations in the present approach.

The pH of silk fibroin aqueous solution was adjusted prior to sonicationin order to determine effects on gelation. Either decreasing orincreasing pH promoted gelation (p<0.05 between * samples in FIG. 3C).The effect of lower pH (pH<4) was more pronounced than the higher pH(pH>9) in inducing gelation (p<0.05 between ⋄ samples in FIG. 3C),consistent with previous studies (Kim et al, 2004; Matsumoto et al.,2006).

Stress/strain curves resulting from mechanical tests on the gelsdisplayed linearity preceding a plateau region, suggesting that the gelshave a large (˜5%-10% strain) and likely viscoelastic characteristic,after which permanent damage is induced by crack formation. The gelsfabricated in this study performed similarly to gels studied in previouswork (Kim et al., 2004), in that the corresponding silk fibroinconcentrations yielded similar values for both yield strength (FIG. 4A)and “traditional” elastic modulus (FIG. 4B). Both metrics appeared to bepositively correlated with silk gel concentration. By inspection, thedifferences in silk fibroin concentration (w/v) were more significantdeterminants of final hydrogel mechanical properties, rather thanvariation due to sonication conditions (FIGS. 4A and 4B). Likewise, theequilibrium modulus values appeared to be positively correlated withsilk gel concentration (FIG. 4C).

When compared with other degradable cell-encapsulating hydrogels, suchas alginate, agarose, polyethylene glycol cross-linked gels, fibrinogenand other systems (Almany & Seliktar 26(15) Biomats. 4023-29 (2005);Kong et al., 24(22) Biomats. 4023-29 (2003); Hung et al., 2004; Bryantet al., 86(7) Biotechnol Bioeng 747-55 (2004); Kang et al. 77(2) J.Biomed. Mater. Res. A 331-39 (2006); Rowley et al., 20(1) Biomats. 45-53(1999); Broderick et al., 72 J. Biomed. Mater. Res. B-Appl Biomater.37-42 (2004); Zhang et al., 15 J. Mater. Sci. Mater. Med. 865-75(2004)), the high-concentration, rapidly forming silk hydrogelsexhibited superior mechanical properties (Table 1). Data were collectedbased on similarities between cell-encapsulation and mechanical testprotocols, in which either “traditional” or equilibrium modulus valueswere determined.

TABLE 1 Comparative mechanical properties among gel systems fromdegradable polymers used for cell encapsulation Material Traditionalmodulus (KPa) Literature Silk Hydrogels 369-1712 Wang et al., 29Biotmats. 1054-64 (2007) Fibrinogen and Fibrinogen-PEG 0.02-4    Almany& Seliktar, 2005 copolymer^(a) Poly(1,8-octanediol citrate) 10.4 Kang etal., 2006 (POC) PEG dimethacrylate-PLA 60-500 Bryant et al., 2004copolymer, (photocross-linked) Gelatin 0.18 Rowley et al., 1999 Gelatin,glutaraldehyde 8.13 Rowley et al., 1999 cross-linked Dex-AI/PNIPAAm5.4-27.7 Zhang et al., 2004 Alginate (calcium-cross- ~25-125  Smith &Mooney, 2003 linked)^(d) Material Equilibrium modulus (KPa) LiteratureSilk Hydrogels 63-441 Wang et al., 29 Biotmats. 1054-64 (2007) Agarose(2% final ~15 Hung et al., 2004 concentration) ^(a)5 mm dia × 5 mmheight. Deformation rate of 1.5 mm/min, modulus based on average slopeof the lower portion of stress-strain curve (<15%). ^(b)6 mm dia × 2.4mm height. Deformation rate of 2 mm/min, modulus based on average slopeof the initial portion of stress-strain curve. ^(c)5 mm dia × 1 mmheight. Load-controlled deformation rate of 40-100 mN/min. ^(d)12.5 mmdia × 1.5 mm height. Load-controlled deformation rate of 25 mN/min,Young's modulus equivalent to the absolute value of the slope obtainedbetween initial preload force 0.01N to 0.25N. ^(e)6 mm dia. Deformationrate of 0.5 mm/min, modulus based on average slope of the lower portionof stress-strain curve. ^(f)12.7 mm dia × 2 mm height. Deformation rateof 1 mm/min. Elastic moduli were obtained from the slope of the stressvs. strain curves, limited to the first 10% of strain. ^(g)Equilibriummodulus calculated from the equilibrium stress and initialcross-sectional area at 10% strain.

Enzymatic (protease XIV) degradation of silk fibroin films, porous solidscaffolds, and silk fibroin yarns have been studied previously (Horan etal., 2005; Kim et al., 2005; Jin et al., 2005). Using the sameconcentration of protease (5 U/mL), all silk fibroin hydrogels showedrapid degradation, with about 80% mass loss in the first four days, witha much slower rate of degradation afterwards (FIG. 5). The degradationof the hydrogels was silk fibroin concentration-dependent. When theconcentration was increased from 4% to 12% (w/v), degradation time toreach 50% mass loss increased from 1.5 days to 3 days (FIG. 5). Thecontrol samples, silk fibroin hydrogels incubated in PBS instead ofprotease, were stable through the incubation period (FIG. 5). The fastdegradation (within days) of silk hydrogels due to proteolytic processesmay be suitable for some applications, such as in wound healingscenarios or rapid drug delivery. It should be noted, however, that theproteolytic degradation times discussed herein are in vitro; in contrastin vivo lifetimes are generally longer and the timeframes will betissue-specific.

hMSCs have been successfully encapsulated in a variety of hydrogelsystems, such as polyethylene glycol, agarose, collagen and alginate,because of the potential of these cells for tissue repair orregeneration and long-term drug release (see Nuttelman et al., 24 MatrixBiol. 208-18 (2005); Nuttelman et al., 27 Biomats. 1377-86 (2006); Maucket al., 14 Osteoarthr. Cartilage 179-89 (2006); Lewus & Nauman, 11Tissue Eng. 1015-22 (2005); Majumdar et al., 185 J. Cell Physiol. 98-106(2000); Boison, 27 Trends Pharmacol. Sci. 652-58 (2006)). Silk hydrogelswith less than 4% (w/v) protein were difficult to manipulate due tophysical limitations. Therefore, for hMSC encapsulation hydrogels of 4%,8%, and 12% (w/v) silk fibroin were used. In all three gelconcentrations, cells retained their original round shape andhomogeneous distribution at day one. At day six, defects appeared onsome cells in the 12% gel and cell morphology had changed. At daytwenty-one, cells in the 4% gel were unchanged when compared with dayone, while cells in the 8% and 12% gels were largely deformed andaggregated. Histological analysis revealed that hMSCs within the matrixof the 4% gel retained round-shape and were nonaggregated throughout thestudy, while those near the surface of the gels grew out of the gel andchanged morphology from round-shape to spindle-like shapes from day six.All hMSCs, either spindle-like near the gel surface or round-shapeencapsulated in the gel, were alive, as seen by green fluorescence inthe live-dead assay. Therefore, hMSCs maintained their activity andfunction in the 4% silk hydrogel system for at least twenty-one days.hMSCs in the 8% and 12% gels, however, largely changed morphology andmany of them died, aggregated and/or dissolved, as seen by the emptycavities in histological images and few green fluorescent spots in thelive-dead assay. The control silk gel, with no cells encapsulated,showed a strong red fluorescence background, which masked the redfluorescence from dead cells in the live-dead assay.

These observations and conclusions were further supported by DNAquantification (PicoGreen assay) (FIG. 6). Cells significantlyproliferated in all three hydrogels over the first 6 days (p<0.05between * samples in FIG. 6). For the 4% gel, cell numbers stoppedincreasing after six days, indicating that maximal gel capacity for cellproliferation was reached. A similar phenomenon was observed in otherhydrogel systems such as PEG and alginate (Nuttleman et al., 2006; Ramdiet al., 207 Exp. Cell Res. 449-54 (1993)). For the 8% and 12% gels, cellnumbers decreased after six days, consistent with the microscopic,histological and live-dead observations. The loss of activity in thehigher concentration gels is likely due to mass transport limitations,but also may be due to mechanical restrictions imposed at these highergel concentrations. The possibility that silk gels were toxic to hMSCscan be excluded because the hMSCs growing on top of the silk gels at 4%,8%, and 12% had growth rates similar to those growing on the controlcell culture plate, and cell morphologies (spindle shape) were similarbetween all groups. Optimization of conditions to stabilize lower gelconcentrations (1% and 2%) may be explored following the teachingsprovided herein, and the diffusion rates of oxygen and nutrients throughvarious concentrations of silk gels may be studied in detail.

A novel method, based on ultrasonication, is provided herein, thatallows the rapid formation of silk fibroin hydrogels. Gelation could beinduced in minutes to hours, depending on the sonication power outputand duration. Gelation was accompanied with β-sheet structure formation,due to changes in hydrophobic hydration. Low concentrations of K⁺ andlow pH accelerated gelation rates, whereas the presence of Ca²⁺ and highconcentrations of K⁺ prevented gelation. The silk fibroin hydrogels hadmechanical properties superior to those reported previously, in therange 369-1712 kPa based on compressive modulus. Gel mechanical strengthincreased with increased silk fibroin solution concentration. The 4%(w/v) silk fibroin hydrogels were suitable for encapsulation for hMSCs;the cells retained viability and proliferation in static cultureconditions over weeks.

The invention will be further characterized by the following exampleswhich are intended to be exemplary of the embodiments.

EXAMPLES Example 1 Silk Fibroin Solutions

Silk fibroin aqueous stock solutions were prepared as previouslydescribed (Sofia et al., 54 J. Biomed. Mater. Res. 139-48 (2001)).Briefly, cocoons of B. mori were boiled for 40 min. in an aqueoussolution of 0.02M sodium carbonate, and then rinsed thoroughly with purewater. After drying, the extracted silk fibroin was dissolved in 9.3MLiBr solution at 60° C. for 4 hours, yielding a 20% (w/v) solution. Thissolution was dialyzed against distilled water using Slide-a-Lyzerdialysis cassettes (MWCO 3,500, Pierce, Rockford, Ill.) for two days toremove the salt. The solution was optically clear after dialysis and wascentrifuged to remove the small amounts of silk aggregates that formedduring the process, usually from environment contaminants that arepresent on the cocoons. The final concentration of silk fibroin aqueoussolution was approximately 8% (w/v). This concentration was determinedby weighing the residual solid of a known volume of solution afterdrying. Silk solutions with lower concentrations were prepared bydiluting the 8% solution with water. To obtain a silk solution withhigher concentration, the 8% solution in a Slide-a-Lyzer dialysiscassettes (MWCO 3,500, Pierce) was dialyzed against 10% (w/v) PEG(10,000 g/mol) solution for at least 24 hours at room temperature (Jin &Kaplan, 2003; Kim et al., 2004). The volume was adjusted with water toreach the desired concentration. All solutions were stored at 4° C.before use.

Example 2 Silk Solutions with Various Salt Concentrations and pH

To determine the effect of salt concentration on silk gelation, KCl andCaCl₂ stock solutions at 1M were added to silk solutions to reach afinal salt concentration of 20 mM to 200 mM. To determine the effect ofpH on gelation, silk solutions were titrated with 1M HCl or NaOHsolutions and the pH was monitored with a pH meter.

Example 3 Screening for Silk Gelation Conditions

To determine silk gelation under various sonication conditions, 0.5 mlof silk (water) solution in a 1.5 ml Eppendorf tube was sonicated with aBranson 450 ultrasonicator (Branson Ultrasonics Co., Danbury, Conn.),which consisted of the Model 450 Power Supply, Converter (Part No.101-135-022), ½″ Externally Threaded Disruptor Horn (Part No.101-147-037), and ⅛″ diameter Tapered Microtip (Part No. 101-148-062).The power output was varied from 10% to 50% amplitude (3 watts-21 watts)and sonication time was varied from 5 sec.-30 sec. To determine theeffects of salts and pH on gelation, 0.5 ml of the silk solutionsprepared as described above were sonicated at 20% amplitude (7 watts)and 15 sec. Solutions were incubated at 37° C. after sonication and thesol-gel transition was monitored visually by turning over the tube andchecking the opacity change of the solution (Matsumoto et al.).

Based on preliminary results, silk fibroin concentrations up to 12%(w/v) were used to maintain lower viscosity, and the 12% solution gelledfaster than the 8% and 4% samples. These results are set forth in Table2, below.

TABLE 2 Gelation time for large volume (5 ml-7 ml) silk fibroin aqueoussolution after sonication. 7 W, 30 s 10 W, 30 s 15 W, 30 s 21 W, 30 s 4%(w/v) No gel in No gel in 5 days 12 hr (1 hr-2 hr 1 week 1 week after2nd sonication) 8% (w/v) 6 day 22-24 hr 45-60 min 15-30 min 12% (w/v)  4day  1.5-2 h 15-30 min gel in tube Note: gelation time was estimated andaveraged based on at least two independent experiments.

Example 4 Circular Dichroism (CD)

A 0.5 ml aliquot of 2% silk (water) solution was sonicated at 20%amplitude (7 watts) for 30 sec., and immediately loaded to a 0.01 mmpath length, sandwich quartz cell (Nova Biotech, El Cajon, Calif.). CDmeasurement was conducted with a Jasco-720 CD spectrophotometer (JascoCo., Japan). All samples were scanned at 37° C. with a 4-s accumulationtime at the rate of 100 nm/min, and the results were averaged from fourrepeated experiments. For the kinetic measurement of silk β-sheetstructure formation, the ellipticity change at 217 nm was monitored for2.5 hours with sampling every 10 sec.

Example 5 Mechanical Testing

A large volume of silk gel was prepared by sonication in order toaccommodate mechanical testing. Silk solutions, 4%, 8%, and 12% (w/v) inglass flasks, were autoclaved 20 min. at 121° C. The autoclaved solutionwas supplemented with sterile Dulbecco's Modified Eagle Medium powder(DMEM powder, Invitrogen, Carlsbad, Calif.) and sodium bicarbonate(Sigma-Aldrich, St. Louis, Mo.) to a concentration of 0.135 g/ml and0.037 g/ml, respectively. The resulting pH of the solution was pH 7.4,which was verified with a pH meter. A 7 ml aliquot was added to a 15 mlFalcon plastic tube and then sonicated at 20%, 30%, 40% amplitude (7watts, 10 watts, 15 watts, respectively) for 30 sec. Six ml of thesonicated solution was added to small culture dishes (BD Falcon™, No.35-3001, BD Biosciences, Palo Alto, Calif.) which were visuallymonitored in a 37° C. incubator, in order to approximate cell cultureparameters, until gelation was complete based on opaque features andcondensation on the gel surface. Subsequently, 9.525 mm diameter plugs(2 mm-3 mm in height) were punched out for mechanical tests immediatelyafter gelation. The gel plugs were pre-conditioned in complete DMEMsolution (Gibco/Invitrogen) for >1 hour prior to testing.

All samples were submerged in DMEM for storage and tested within 24hours. Samples were evaluated on a 3366 Instron machine (Norwood, Mass.)equipped with unconfined compression platens and a 100N load transducer.The compressive extension method was employed with 1 mm/min rate ofextension. The compressive stress and strain were determined and theelastic modulus was calculated based on a semi-automatic technique. Thestress-strain diagram was segmented into eight sections below a cut-offstress level set beyond the initial linear portion of the diagram. Usingleast-squares' fitting, the highest slope among these eight sections wasdefined as the compressive modulus for the sample. The compressivestrength was determined using an offset-yield approach. A line was drawnparallel to the modulus line, but offset by 0.5% of the sample gaugelength. The corresponding stress value at which the offset line crossedthe stress-strain curve was defined as the compressive strength of thescaffold. This testing was performed according to a modification basedon the ASTM method F451-95.

Two unconfined compression testing regimes were pursued to evaluate theinfluence of sonication conditions on mechanical performance. First,strain-to-failure test was used to extract a traditional materialstiffness property and to observe a failure response (Almany & Seliktar26(15) Biomats. 2467-77 (2005); Kong et al., 24(22) Biomats. 4023-29(2003)). Second, a stress relaxation test was used to evaluateequilibrium modulus properties, based on test parameters of Hung et al.(32 Ann. Biomed. Eng. 35-49 (2004)). Together, these measures providebroad comparisons against the published properties of other degradablehydrogels used for cell encapsulation. N=4 samples were evaluated forevery group reported and were tested on a 3366 Instron machine (Norwood,Mass.) equipped with unconfined compression platens and 100 N loadtransducer and sample data exported using Bluehill Software Version 2.0.

For strain-to-failure testing, each sample was compressed at anextension-controlled rate of 1 mm/min, beginning after nominal tareloads were reached and sample heights recorded. The compressive stressand strain were determined by normalizing against sample geometries andthe “traditional” elastic modulus was calculated as the slope of atangency line established at the 5% strain portion of each stress/straincurve. The yield strength was determined by offsetting a line parallelto the tangency line by 2% strain; where the offset line intersected thestress/strain response was defined as the yield strength (whichcoincided with failure onset). For stress relaxation testing, sampleswere submerged in phosphate-buffered saline (PBS) and left under anominal tare load for 200 s. Thereafter, samples were compressed at 1mm/s until 10% strain was reached, which was held for 20 min. Theequilibrium modulus was calculated by normalizing the relaxation stressby 10% strain.

Example 6 In Vitro Enzymatic Degradation of Silk Gels

Silk gel plugs (diameter=4 mm; height=2 mm-3 mm) at 4%, 8%, 12% (w/v)were prepared as described above and then immersed in 1 mL of ProteaseXIV (Sigma-Aldrich) solution in a 24-well plate. The protease solutionwas freshly prepared by dissolving the enzyme powder in PBS to reach aconcentration of 5 U/mL and replaced with newly prepared solution every24 hr. The control plugs were immersed in 1 mL of PBS which was alsorefreshed every 24 hr. All samples were incubated at 37° C. At days 1,2, 3, 4 and 7, four plugs were washed with water, wiped with tissuepaper to remove excess water on the gel surface, and weighed.

Example 7 hMSCs Seeding and Culturing in Silk Gels

hMSCs were isolated from fresh whole bone marrow aspirates fromconsenting donors (Clonetic-Poietics, Walkersville, Md.) as describedpreviously (Meinel et al., 71 J. Biomed. Mater. Res. A 25-34 (2004);Meinel et al., 88 Biotechnol. Bioeng. 379-91 (2004)), and cultureexpanded in a growth medium containing 90% DMEM, 10% fetal bovine serum(FBS), 0.1 mM non-essential amino acids, 100 U/mL penicillin, 1000 U/mLstreptomycin, 0.2% fungizone antimycotic, and 1 ng/mL basic fibroblastgrowth factor (bFGF). Before use, passage 3-4 cells were trypsinizedfrom culture flasks and resuspended in DMEM to obtain a cell density of5×10⁷ cell/mL. Fifteen mL of silk solution at 4%, 8%, and 12% (w/v) weresteam sterilized (autoclaved) and supplemented with DMEM powder andsodium bicarbonate as described above. An aliquot of 5 mL was added to a15-mL falcon plastic tube and a total of two tubes (control and cellseeded) were prepared for each silk concentration. A 4% (w/v) silksolution (5 mL) was sonicated in a laminar flow hood at 50% amplitudefor 30 sec, and after 30 min incubation the solution was sonicated againunder the same conditions. After the second sonication, the solution wascooled to room temperature within 5 min-10 min, and then 50 mL of thecell suspension was added and mixed with the sonicated silk solution toreach a final concentration of 5×10⁵ cells/mL. The control sample wassonicated in the same way, but 50 mL of DMEM was added instead of thecell suspension after the sonication. An aliquot of 1.5 mL of themixtures was quickly pipetted into 12-well cell culture plates, with atotal of three wells prepared for each sample group. The 8% and 12%(w/v) solutions were sonicated once at 40% and 30% amplitude,respectively, for 30 s. A 50 ml aliquot of hMSC suspension was added andthe mixture was plated as described above. All plates were thenincubated at 37° C. and 5% CO₂.

Once the silk gelled in the plates within 0.5 hr-2 hr, small plugs(diameter=4 mm; height=2-3 mm) were punched out of the gels and placedin the wells of a new 24-well plate. The plugs were then cultured in 1mL of growth medium containing 90% DMEM, 10% FBS, 0.1 mM nonessentialamino acids, 100 U/mL penicillin, 1000 U/mL streptomycin, 0.2% fungizoneantimycotic at 37° C. and 5% CO₂. For microscopy imaging, the hMSCencapsulated silk gels with a volume of 0.5 mL were prepared in 24-wellplates and cultured in 1 mL of the same growth medium and under the sameconditions as above, and images were taken at desired time points.

Example 8 Analyses of hMSCs Encapsulated in Silk Gels

Phase contrast microscopy—At days 2, 6, 14 and 21 of culture, cellmorphology was monitored by a phase contrast light microscopy (CarlZeiss, Jena, Germany) equipped with a Sony Exwave HAD 3CCD color videocamera.

Cell proliferation—Cell proliferation was assessed by DNA assay.Briefly, at each time point, 4 gel plugs from each group were washedwith PBS, pH 7.4, weighed (wet weight), and chopped with microscissorsin ice. DNA content (N=4) was measured using PicoGreen assay (MolecularProbes, Eugene, Oreg.), according to the manufacturer's instructions.Samples were measured fluorometrically at an excitation wavelength of480 nm and an emission wavelength of 528 nm. DNA content was calculatedbased on a standard curve obtained in the same assay, and furthernormalized by the wet weight of each gel plug.

Cell viability: the viability of the hMSCs in the gel plugs was examinedby a live/dead assay (Molecular Probes, Eugene, Oreg.). Briefly, at theend of culture, a gel plug of each group seeded with hMSCs were washedwith PBS, cut into two halves, and incubated in 2 mM calcein AM(staining live cells) and 4 mM ethidium homodimer (EthD-1, staining deadcells) in PBS for 30 min at 37° C. The cross-section of the cut gel wasimaged by Confocal microscopy (Bio-Rad MRC 1024, Hercules, Calif.) withLasersharp 2000 software (excitation/emission ˜495 nm/˜515 nm). Depthprojection micrographs were obtained from a series of horizontalsections, imaged at various distances from each other (1 μm-10 μmincrements), based on the total height of a well-defined cell colony.Still images at various depths were captured and a series of micrographswere later combined for “z-stacked” compilation images.

Histology. Silk gels seeded with cells were washed in PBS and fixed in10% neutral-buffered formalin for 2 days before histological analysis.Samples were dehydrated through a series of graded ethanols, embedded inparaffin and sectioned at 5 mm thickness. For histological evaluation,sections were deparaffinized, rehydrated through a series of gradedethanols, and stained with hematoxylin and eosin (H&E).

Example 9 Statistics

Statistical analyses were performed using the Student's t-test.Differences were considered significant when pp 0.05 and highlysignificant when pp 0.01.

1. A process of rapidly forming silk fibroin gelation, comprisingexposing silk fibroin to a treatment comprising ultrasonication for aperiod of about 5 seconds to about 60 seconds to initiate gelation,wherein substantial silk fibroin gelation forms less than 24 hours afterthe ultrasonication treatment, and wherein the silk fibroin is in theform of an aqueous solution having a pH of 7.5 or higher.
 2. The processof claim 1, wherein the silk fibroin gelation forms less than two hoursafter the ultrasonication treatment.
 3. The process of claim 1, whereinthe silk fibroin undergoes gelation at a time period ranging from aboutfive minutes to about two hours after the ultrasonication treatment. 4.The process of claim 1, wherein the treatment further comprises a saltsolution.
 5. The process of claim 4, wherein the salt solution comprisesions selected from the group consisting of potassium, calcium, sodium,magnesium, copper, zinc, and combinations thereof.
 6. The process ofclaim 5, wherein the salt is potassium, the salt concentration is lessthan 100 mM.
 7. A method of controlling gelation time of silk fibroin bycontacting a silk fibroin solution with an ultrasonication treatment forperiod of about 5 seconds to about 60 seconds to initiate gelation,wherein the silk fibroin undergoes substantial gelation within about twohours, and wherein the silk fibroin solution has a pH of 7.5 or higher.8. The method of claim 7, wherein the silk fibroin undergoes gelation ata time period ranging from about five minutes to about two hours afterthe ultrasonication treatment.
 9. The method of claim 7, wherein thegelation time is controlled through the amplitude of the ultrasonicationand the concentration of the silk fibroin solution.
 10. The method ofclaim 7, wherein the treatment further comprises a salt solution. 11.The method of claim 10, wherein the gelation time is controlled throughthe concentration of the silk fibroin solution and the concentration ofthe salt solution.
 12. The method of claim 11, wherein the concentrationof the silk fibroin is 4 wt % or lower, the salt solution comprisespotassium ions, and the concentration of the potassium salt solutionranges from 20 mM to 100 mM.
 13. The method of claim 10, wherein thegelation time is controlled through the concentration and the pH of thesalt solution.
 14. The method of claim 13, wherein the salt solutioncomprises potassium ions, the concentration of the potassium saltsolution ranges from 20 mM to 100 mM.
 15. A method of encapsulating atleast one agent in silk fibroin, comprising: a. contacting a silkfibroin solution with an ultrasonication treatment for a period of about5 seconds to about 60 seconds to initiate gelation, wherein the silkfibroin solution has a pH of 7.5 or higher; and b. introducing theagent(s) to the silk fibroin solution before substantial gelation occursin the silk fibroin solution; c. to form a silk-fibroin encapsulatedagent.
 16. The method of claim 15, wherein the agent is a therapeuticagent or a biological material, or both.
 17. The method of claim 16,wherein the agent is at least one biological material selected from thegroup consisting of cells, proteins, peptides, nucleic acids, PNA,aptamers, antibodies, hormones, growth factors, cytokines, enzymes,antimicrobial compounds, and combinations thereof.
 18. The method ofclaim 17, wherein said cell is a stem cell.
 19. The method of claim 17,wherein a cell growth medium is introduced into silk fibroin with thebiological material.
 20. The method of claim 16, wherein the agent is atherapeutic agent selected from the group consisting of small molecules,drugs, and combinations thereof.
 21. The method of claim 15, wherein thesilk-fibroin encapsulated biological material is suitable for abiodelivery device.
 22. The method of claim 15, wherein substantialgelation occurs within about 2 hours.
 23. The method of claim 15,wherein substantial gelation occurs in the time period ranging fromabout five minutes to about two hours.
 24. The process of claim 15,wherein the treatment further comprises a salt solution.
 25. A method ofencapsulating at least one agent in silk fibroin, comprising: a.introducing the agent(s) to a silk fibroin solution, wherein the silkfibroin solution has a pH of 7.5 or higher; and b. contacting a silkfibroin solution with an ultrasonication treatment for a period of about5 seconds to about 60 seconds to initiate gelation; c. to form asilk-fibroin encapsulated agent.
 26. The method of claim 25, wherein theagent is a therapeutic agent selected from the group consisting of smallmolecules, drugs, and combinations thereof.
 27. The method of claim 25,wherein substantial gelation occurs within about two hours.
 28. Themethod of claim 25, wherein substantial gelation occurs in the timeperiod ranging from about five minutes to about two hours.
 29. Themethod of claim 25, wherein power of ultrasonic waves is 3 watts to 21watts.
 30. The method of claim 15, wherein power of ultrasonic waves is3 watts to 21 watts.
 31. The method of claim 7, wherein power ofultrasonic waves is 3 watts to 21 watts.
 32. The method of claim 1,wherein power of ultrasonic waves is 3 watts to 21 watts.